KR20140048107A - Method for preparing multiple antigen glycopeptide carbohydrate conjugates - Google Patents

Abstract

The present invention relates to a method for preparing a carbohydrate T cell epitope conjugate of formula (I):
M (TB) _n (I)
In the above formula, M, T, B and n are as defined in claim 1.

Description

METHOD FOR PREPARING MULTIPLE ANTIGEN GLYCOPEPTIDE CARBOHYDRATE CONJUGATES}

The present invention relates to a method for preparing a carbohydrate T cell epitope conjugate of formula (I), and to a carbohydrate T cell epitope intermediate of formula (II) useful according to the method.

Over the past decade, a new class of synthetic immunogens, MAG-Tn3, has been developed that present tumor-associated Tn antigens. MAG-Tn3 is a fully synthetic glycopeptide (MW = 10'897Da) that binds a carbohydrate Tn antigen (as a tri-Tn cluster) to a peptide CD4 ⁺ T cell epitope on a tetravalent backbone. [5, 9].

MAG-Tn3 has the following structure [S (α-D-GalNAc) -T (α-D-GalNAc) -T (α-D-GalNAc) -QY ⁵ IKANS ¹⁰ KFIGI ¹⁵ TEL] ₄ -K ₂ -K- matches β-Ala:

MAG stands for Multiple Antigen Glycopeptide.

Thus, MAG-Tn3 is a carbohydrate peptide conjugate B4-T4-M, consistent with the formula:

In the above formulas,

KKK is the dendritic polyLysine core (M),

T is a peptide CD4 ⁺ T cell epitope having the sequence of QYIKANSKFIGITEL,

Tn3 is a tri-Tn B cell epitope having the sequence (α-GalNAc) Ser- (α-GalNAc) Thr- (α-GalNAc) Thr.

Based on successful in vivo results obtained in mice and primates [8, 9], MAG-Tn3 is a good therapeutic vaccine candidate for treating carcinomas that require entry into phase I / II clinical trials.

WO9843677-Multiple antigen glycopeptide carbohydrate, vaccine comprising the same and use specifically

Kaiser, E., R. L. Colescott, C. D. Bossinger, and P.I. Cook, Anal Biochem (1970). 34: 595-8. Maemura, M., A. Ohgaki, Y. Nakahara, H. Hojo, and Y. Nakahara, Bioscience Biotechnology and Biochemistry (2005). 69: 1575-1583. Tam, J.P., W.F. Heath, and R.B. Merrifield, J Am Chem Soc (1986). 108: 5242-5251. Tanaka, E., Y. Nakahara, Y. Kuroda, Y. Takano, N. Kojima, H. Hojo, and Y. Nakahara, Bioscience Biotechnology and Biochemistry (2006). 70: 2515-2522. Bay, S., R. Lo-Man, E. Osinaga, H. Nakada, C. Leclerc, and D. Cantacuzene, J. Peptide Res. (1997). 49: 620-625; Fmoc solid phase peptide synthesis, A practical approach, Edited by W.C. Chan and P. D. White, Oxford University Press. Sander, J. and H. Waldmann, Chem Eur J (2000). 6: 1564-1577. Lo-Man, R., S. Vichier-Guerre, S. Bay, E. Deriaud, D. Cantacuzene, and C. Leclerc, J. Immunol. (2001). 166: 2849-2854. Lo-Man, R., S. Vichier-Guerre, R. Perraut, E. Deriaud, V. Huteau, L. Ben Mohamed, O.M. Diop, P. O. Livingston, S. Bay, and C. Leclerc, Cancer Res (2004). 64: 4987-4994 Babino, A., D. Tello, A. Rojas, S. Bay, E. Osinaga, and P.M. Alzari, FEBS Lett. (2003). 536: 106-110.

Synthetic routes for preparing peptide carbohydrate conjugates, such as MAG-Tn3, in particular, are disclosed in International Patent Application WO 98/43677 and US Pat. No. 6,676,946. In this process, carried out on a small scale (1-10 mg final compound), the carbohydrate Tn3 antigen is included in the dendrimer peptide M (T) ₄ building block. It should be noted that the inclusion of the Tn3 antigen is achieved by fully unprotected saccharides which may seem advantageous to avoid the final deprotection step.

However, the inventors have shown that this method fails when scaled up. In fact, extra-incorporation of Tn residues is difficult to control and affects crude purity and overall yield. Furthermore, purification of such high molecular weight glycopeptides is complex.

Thus, there is a need for an improved method of manufacturing MAG-Tn3 that overcomes the problems of the prior art, in particular in a repeatable manner, in particular on a larger scale, which makes it possible to obtain better yields and purity.

Thus, in one aspect, the present invention provides MAG-Tn3, and more generally carbohydrate T cell epitopes of Formula (I) (M (TB) _n ), which allows for large scale production, with better yield and purity. Provides a novel process for making a conjugate:

M(T-B_Pr)_n, (II)

M(T-B)_n, (I)nB _Pr + M (T) _n

M (TB _Pr ) _n, (II)

M (TB) _n , (I)

The method according to the invention advantageously makes it possible to minimize byproduct synthesis, thereby improving the robustness of the process and making it possible to scale up the synthesis in a repeatable manner.

More specifically, protecting the hydroxyl group of the carbohydrate B cell epitope with an appropriate protecting group (Pr) before incorporating the B cell epitope into the M (T) _n building block provides better control of B cell epitope inclusion. Has been found to improve both yield and purity, particularly at large scale.

Another object of the present invention is a novel carbohydrate T cell epi of formula (II) (M (TB _Pr ) _n ), which is a compound useful for the preparation of carbohydrate T cell epitope conjugates of formula M (TB) _n with high yield and purity. It is to provide a saw assembly.

A further object of the present invention is to provide a carbohydrate T cell epitope conjugate of formula (I) (M (TB) _n ) having a purity grade better than 95%, obtainable by the process according to the invention.

These and other objects, features and advantages of the method according to the invention will be disclosed in the following detailed description.

Accordingly, in one aspect, the present invention relates to a method for preparing a carbohydrate T cell epitope conjugate of formula (I):

M (TB) _n (I)

In the above formulas,

M is a dendrimer polylysine core;

T is a T cell epitope comprising a peptide;

B is a carbohydrate B cell epitope comprising one or more carbohydrate residues (b);

n is an integer indicating the number of -T-B groups covalently bonded to M;

The method comprises the steps of:

i) A carbohydrate T cell epitope conjugate M by combining a protected carbohydrate B cell epitope (B _Pr ) with compound M (T) _n , _wherein the hydroxyl group of the carbohydrate moiety (b) is protected with a protecting group (Pr). Forming (T-B _Pr ) _n ,

The protecting group (Pr) is allyl, p -methoxybenzyl (PMB), t-butyldimethylsilyl (TBDMS), benzyloxymethyl (BOM), levulinyl (Lev), benzoyl (Bz), 2,5 -difluoro-benzoyl, chloroacetyl, attached to the benzyl (Bn) or or selected from the group consisting of acetyl (Ac), C ₅ -C ₆ isopropylidene ketal (isopropylidene ketal), or C ₅ -C ₆ cyclic alkyl carbonate Formed with two hydroxyl groups

And

ii) to give the conjugate M (TB _Pr) Carbohydrate T cells by removing the protecting groups Pr from the _n-epi junction body top M (TB) to obtain a _n.

Dendrimer Polylysine Core (M)

The polylysine centers of the conjugates of formula (I) are dendrimer structures which may be represented in a star shape, having a plurality of branches (-T-B), which may be the same or different from one another.

These branches are covalently linked to the NH ₂ terminus of each lysine residue of the dendrimer center, in particular by peptide bonds -NH-C (= 0)-.

The valence m of the dendrimer polylysine core (M), that is, the number of NH ₂ termini of each lysine residue is m ≥ n, preferably m = n, where n is the number of (-TB) branches covalently bonded to M Specifies.

In another aspect, the number n of (-T-B) branches reaches 4 to 16, in particular 4 to 8.

In a further aspect, the dendrimer polylysine center M comprises at least 3 lysine residues, in particular 3 to 15 lysine residues, more particularly 3 to 7 lysine residues.

In a further aspect, the M (TB) _n conjugate is selected from formula (la) or (lb):

In the above formulas,

K is a lysine residue,

T and B are as defined above.

Advantageously, the dendrimer structures (Ia) and (Ib) provide high density antigens on the surface of the dendrimer polylysine central M.

In a further aspect, M is (K) ₂ K-βAla-OH of the formula:

(T-B) eggplant

The presence of both carbohydrate B cell epitopes and T cell epitopes on the conjugate of formula (I) makes the conjugate of formula (I) an efficient immunogen.

T cell epitope

As used herein, T cell epitope refers to peptide properties that can induce antigens, particularly T cell responses.

Such epitopes are described in particular in S. Stevanovic, " Identification of Tumour-associated T-cell epitopes for vaccine development" , Nature Reviews, Vol. 2, July 2002, p. 1 to 7; JH Kessler, CJM Melief, "Identification of T-cell epitopes for cancer immunotherapy", Leukemia (2007) 21, 1859-1874, or Cancer immunity / peptide database website [http: / /www.cancerimmunity.org/peptide database / tumorspecific.htm].

The T cell epitopes included in the conjugates of formula (I) may be the same or different and may not be peptides or peptides.

In certain aspects, the carbohydrate T cell epitope conjugate is a carbohydrate peptide conjugate. Peptide T cell epitopes may comprise from 2 to 50 amino acids.

The T cell epitope may in particular be selected from CD8 ⁺ , or CD4 ⁺ T cell epitopes.

CD8 ⁺ T cell epitopes recognized as tumor markers may be selected from the group consisting of:

-MUC-1 peptide (pancreas, breast)

MAGE 1 and 3 (melanoma, lung) (T. Boon et al. (1995), Immunology Today, vol. 16 n ° 7, pp 334-336)

-pme117 / gp 100 (melanoma)

Tyrosinase (melanoma) BAGE (melanoma)

-GAGE (melanoma) LB-33-B (melanoma)

-CDK4

p185 ^HER (breast, ovary)

CEA MART1 / Melan-A (melanoma)

Or in A. Van Pel et al. (1995) Immunological. Reviews n ° 145, pp 229-250 or P. G. Coulie (1995), Stem Cells, 13, pp 393-403.

In certain aspects, T can be or include a CD4 ⁺ T cell epitope, which can be, in particular, a poliovirus (PV) protein fragment, tetanus toxin fragment or PADRE peptide.

As an example of a CD4 ⁺ T cell epitope selected among poliovirus (PV) protein fragments, a synthetic peptide (KLFAVWKITYKDT) (SEQ ID N ° 4) that matches the 103-115 sequence of VP1 protein from poliovirus type 1 will be mentioned. Can be.

As an example of a CD4 ⁺ T cell epitope selected from tetanus toxin fragments, the following fragments may be mentioned:

830-844 sequence of tetanus toxin (QYIKANSKFIGITEL) (SEQ ID N ° 1)

947-967 sequence of tetanus toxin (FNNFTVSFWLRVPKVSASHLE) (SEQ ID N ° 2)

1273-1284 sequence of tetanus toxin (GQIGNDPNRDIL) (SEQ ID N ° 3).

These peptide T cell epitopes generally bind to a plurality of class II human and murine Major Histocompatibility Complex (MHC) molecules, resulting in a CD4 + T cell response, which is associated with polymorphism of MHC molecules present between individuals. It avoids the restriction problem. Moreover, the use of tetanus toxin peptides will increase the immunogenicity of antigens present in the conjugates of the invention as a result of vaccination of many individuals with tetanus toxoids.

As further examples of peptide T cell epitopes, see S. Stevanovic, " Identification of Tumour-associated T-cell epitopes for vaccine development" , Nature Reviews, Vol. 2, July 2002, p. 1 to 7; JH Kessler, CJM Melief, "Identification of T-cell epitopes for cancer immunotherapy", Leukemia (2007) 21, 1859-1874], or Cancer Immunity / Peptide Database Website: http://www.cancerimmunity.org/peptide database Reference may be made to /tumorspecific.htm.

Examples of nonpeptide T cell epitopes may include, inter alia:

-C. Fragments of pneumococcal type 4 polysaccharides, and oligosaccharide tetanus toxin conjugates, described in C. A. M. Peeters (1991), The Journal of Immunology, 146, 4309-4314.

-A. F. M. Verheul (1991), Detection and Immunity, vol. 59, n ° 10, pp. Meningococcal liposaccharides described in 3566-3573.

B cell epitope

As used herein, B cell epitope means an antigen capable of eliciting a B cell response.

As used herein, carbohydrate means sugars, in particular monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

In a preferred aspect, the carbohydrate moiety (b) forming the B cell epitope of the conjugate of formula (I) is an N-acetylgalactopyranosyl residue, or derivative thereof.

In certain aspects, the carbohydrate residue (b) is attached to an amino acid, peptide, or lipid residue. In a still further aspect, the carbohydrate moiety is an O-glycosyl amino acid or peptide. In a further aspect, the B cell epitope is attached to the dendrimer structure M (T) _n via the amino acid or peptide.

The B cell epitope may comprise one or more carbohydrate residues (b), in particular 1 to 10, in particular 1 to 6 carbohydrate residues.

Such B cell epitopes may be selected from tumor (cancer) glycosidic antigens, in particular:

Acidic glycolipids such as, for example, ganglioside GD2, GD3 and GM3 (melanoma) and, for example, Lewisy, Ley (breast, prostate, ovary) and globo H (Globo H). Glycolipid classes, including neutral glycolipids such as H) (breast, prostate, ovary) antigens;

For example, Tn antigens (α-GalNAc-Ser or α-GalNAc-Thr), TF antigens (β-Gal- (1-3) -α-, two tumor markers that are common in carcinomas but usually not in normal tissues) GalNAc-Ser or β-Gal- (1-3) -α-GalNAc-Thr (Springer GF Science 224,1198-1206 (1984)) (ovary, breast, lung), or di-Tn (α O-glycosyl peptide (or amino acid) class such as -GalNAc-Ser / Thr) ₂ , tri-Tn (α-GalNAc-Ser / Thr) ₃ or hexa-Tn (α-GalNAc-Ser / Thr) ₆ .

B cell epitopes of the conjugates according to the invention can also be used, for example, capsular bacterial polysaccharides of the Neisseria meningitis , Haemophilus influenzae , Streptococcus pneumoniae and Streptococcus group. bacterial polysaccharides).

Polysaccharides are carbohydrate residues obtained from synthetic processes.

The B cell epitope of the conjugate may also be of fungal origin, for example as isolated from yeast Saccharomyces.

In a preferred aspect, the B cell epitope of the conjugate of formula (I) is preferably a tumor marker such as, for example, Tn and TF antigens.

The tumor marker may be selected from the group comprising Tn, di-Tn, tri-Tn (Tn3), hexa-Tn (Tn6), or TF antigen.

In a further aspect B is or comprises a carbohydrate moiety selected from the group consisting of:

α-GalNAc,

α-GalNAc-Ser,

α-GalNAc-Thr,

β-GalNAc,

β-GalNAc-Ser,

β-GalNAc-Thr,

β-Gal- (1-3) -α-GalNAc-Ser,

β-Gal- (1-3) -α-GalNAc-Thr,

(α-GalNAc-Ser / Thr) ₂ ,

(α-GalNAc-Ser / Thr) ₃ , and

(α-GalNAc-Ser / Thr) ₆ ,

In a preferred aspect, B is or comprises (α-GalNAc-Ser / Thr) ₃ , most preferably (α-GalNAc) Ser- (α-GalNAc) Thr- (α-GalNAc) Thr residues.

In another preferred embodiment, the conjugate of formula (I) is MAG-Tn3.

i) step

The method according to the invention comprises: i) a carbohydrate by combining a protected carbohydrate B cell epitope (B _Pr ) with compound M (T) _n , _wherein the hydroxyl group of the carbohydrate moiety (b) is protected with a protecting group (Pr). Forming a T cell epitope conjugate M (T-B _Pr ) _n ,

The protecting group (Pr) is allyl, p -methoxybenzyl (PMB), t-butyldimethylsilyl (TBDMS), benzyloxymethyl (BOM), levulinyl (Lev), benzoyl (Bz), 2,5-di Fluorobenzoyl, chloroacetyl, benzyl (Bn) or acetyl (Ac), or

Forming two hydroxyl groups attached to a C ₅ -C ₆ isopropylidene ketal or a C ₅ -C ₆ cyclic alkylcarbonate.

In a preferred aspect, Pr is benzyl (Bn) or acetyl (Ac).

In a preferred aspect, step i) comprises the following steps:

a) a carbohydrate T cell by combining a first protected carbohydrate moiety (b _Pr ) with compound M (T) _n , wherein the first protected carbohydrate moiety (b _Pr ), in particular the hydroxyl group protected with a protecting group (Pr) selected from benzyl (Bn) or acetyl (Ac) Forming an epitope conjugate M (Tb _Pr ) _n ; And optionally

b) repeating step a) with additional protected carbohydrate moiety (b _Pr ) until a protected carbohydrate conjugate of formula (II) M (TB _Pr ) _n is obtained.

Advantageously, it has been demonstrated that this protecting group Pr enables binding control of each carbohydrate residue b, or B cell epitope B with M (T) _n in step i). In addition, it has been shown that the removal of such protecting groups makes it possible to obtain the desired product with improved compromise between yield and purity.

The hydroxyl group of the carbohydrate moiety can be protected according to conventional methods, with the aforementioned protecting groups (Pr), in particular benzyl or acetyl groups.

Protected B cell epitope B _Pr or carbohydrate residues (b _Pr ) can be prepared from commercially available or commercially available starting materials and / or according to conventional methods.

In a preferred aspect, the dendrimer polylysine core M, and thus the subsequent M (T) _n building blocks, are anchored to the solid support, thereby allowing repeat solid phase peptide synthesis.

As an example of a solid support, a polystyrene resin or Fmoc-β-Ala-TentaGel R Trt functionalized with p -benzyloxybenzyl alcohol (Wang resin), which is then conjugated with a Fmoc-β-Ala- group Those sold may be mentioned. The M center may in particular be attached via a β-Ala-OH moiety. The resin substitution ratio, ie, the bonding ratio of the resin by the Fmoc-β-Ala- group, may range from 0.2 to 0.05 mmol / g, preferably 0.10 to 0.13 mmol / g.

In certain aspects, b _Pr is a protected O-glycosyl amino acid or peptide. Protected O-glycosyl peptides can be linked by successive introduction of protected structural O-glycosyl amino acid residues b _Pr to the M (T) _n building blocks.

In this regard, solid phase peptide and glycopeptide synthesis can be performed using standard Fmoc chemistry protocols [5] and [6]. N-α-Fmoc amino acids and glycosylated amino acids or peptides are stepwise included in the peptide chain.

Thus, step i) can be carried out by reacting the first protected N-α-Fmoc O-glycosyl amino acid b _Pr with the M (T) _n building block. More specifically, the carbo group (COOH) of the first O-glycosyl amino acid b _Pr reacts with each of the two T ₂ NH ₂ forms to form the peptide “bond” (—C (═O) NH—).

Fmoc is cleaved, for example, in the presence of 20% piperidine in DMF or NMP. The second protected N-α-Fmoc O-glycosyl amino acid b _Pr may then react similarly to the NH ₂ group of the first protected amino acid residue.

Thus, if the B cell epitope comprises a plurality of carbohydrate residues (b), step i) of the method according to the invention will result in the binding according to step a) to obtain a carbohydrate T cell epitope conjugate M (TB _Pr ) _n . It may include repeating until.

These peptide bonds can be performed in the presence of binding reagents such as HATU and DIPEA or DIC / HOBt, and PyBOP in a polar aprotic solvent such as DMF or NMP.

ii) step

In certain aspects, Pr is benzyl. In this case, deprotection step ii) can be carried out in the presence of TfOH or by catalytic hydrogenation.

In certain aspects, step ii) is catalytic hydrogenation.

In a preferred aspect, catalytic hydrogenation is carried out in the presence of Pd / C, in particular 10% Pd / C (% w / w), as catalyst. The weight ratio of the conjugate / catalyst of formula (II) may vary from 10/2 to 10/10, preferably about 10/8. The catalyst can be added in small amounts over a long period of time.

Catalytic hydrogenation is preferably carried out in NMP / H ₂ O as solvent, in particular in a volume ratio of 87.5 / 12.5.

The catalytic hydrogenation reaction is preferably carried out at temperatures up to 20-40 ° C., in particular at about 37 ° C.

The catalytic hydrogenation reaction is preferably carried out under pressures ranging from 1 to 10 bar, more preferably about 5 bar.

In another aspect, step ii) is performed in the presence of TfOH.

Preferably, the conjugate of formula (II) reacts with TfOH in the presence of TFA, DMS and m -cresol. The relative ratio of TfOH / TFA / DMF / m-cresol may be 1/5/3/1 v / v / v / v.

In another aspect, the protecting group Pr is acetyl.

Deprotection of the acetyl protecting group in step ii) is preferably carried out in the presence of hydrazine, in a protonic polar solvent, for example in alcohol such as methanol. This reaction can be carried out at room temperature, ie between 15 and 25 ° C.

The molar ratio of hydrazine relative to the compound of formula (II) may vary from 100 to 1500 molar equivalents.

Deprotection of acetyl can alternatively be carried out in the presence of MeONa as solvent, in particular in MeOH, at room temperature, ie between 15 and 25 ° C.

In certain aspects, once immobilized on the solid support, the conjugate of formula (II) obtained at the end of i) is preliminarily cleaved from the solid support before the ii) deprotection step is performed. Such cleavage can be carried out in the presence of TFA and TIS in water, for example at the following volume ratio 95 / 2.5 / 2.5 v / v / v.

iii) step

The process according to the invention may additionally comprise one or more purification steps, in particular iii) steps following step ii), consisting of reverse phase high performance liquid chromatography (RP-HPLC).

iv) step

The process according to the invention may additionally comprise a subsequent step iv) of recovering the product.

M (T) _n  synthesis

In certain aspects, the method according to the invention may further comprise preparing a conjugate M (T) _n .

In certain embodiments, conjugate M (T) _n is prepared by stepwise introduction of N-protected amino acid residues that make up the peptide T cell epitope starting from the dendrimer polylysine core. N-protected amino acid residues are in particular Fmoc amino acid residues.

Amino acid binding can be performed in the presence of one or more peptide binding reagents such as DIC, HOBt, PyBOP, HATU or DIPEA in a polar aprotic solvent such as DMF. Particular preference is given to combinations (DIC / HOBt and PyBOP) or alternatives (HATU / DIPEA). Alternative binding reagents are also described in E. Valeur; M. Bradley, Chem Soc Rev (2009) 38, 606; A. El-Faham et al. Chem Eur J (2009) 15, 9404 or R. Subiros-Funosas et al. Org Biomol Chem (2010) 8, 3665.

Following each peptide bond, the N-amino acid protecting group is removed. By way of example, the Fmoc protecting group can be removed in the presence of piperidine in a polar aprotic solvent such as DMF.

Amino acid residues AA ^9-10 and AA ^15-16 can be included as pseudo-proline dipeptides in connection with the synthesis of peptide fragment SEQ ID n ° 1. Advantageously, the inclusion of such dipeptides has demonstrated a significant impact on the crude quality of the product.

M synthetic

In certain aspects, the method according to the invention further comprises the step of preparing the dendrimer polylysine core M.

Dendrimer polylysine core M can be prepared according to the method disclosed in US Pat. No. 6,676,946.

Carbohydrate T Cell Epitope Conjugate M (T-B) _n

In another aspect, the present invention advantageously provides a carbohydrate T cell epitope conjugate M (TB) _n having a purity ≧ 95% grade obtainable according to the method of the present invention.

This purity grade can be obtained by performing purification step (iii) after step (ii), in particular reverse phase high performance liquid chromatography (RP-HPLC). By way of example, RP-HPLC has a low-granulometry column, in particular having a granulometry of less than 15 μm, in particular less than about 10 μm or less than 5 μm and / or less than or equal to about 300 μs, especially less than 200 μs, especially 100 μs It may be performed with less than one pore size. The stationary phase may be a reverse phase based on silica gel (also called C18 silica) bonded to octadecyl groups. Elution can be performed with a shallow gradient, for example, from purified water (0.1% TFA) / acetonitrile from 70/30 to 60/40 over a period of about 20 minutes.

The purity grade of the conjugate of formula (I) can be determined by any conventional method, in particular by RP-HPLC.

The purity is preferably ≧ 96%, in particular ≧ 98%, advantageously ≧ 99%.

Carbohydrate T Cell Epitope Conjugate M (T-B _Pr ) _n

In a further aspect, the present invention provides a carbohydrate T cell epitope conjugate of formula (II):

M (TB _Pr ) _n (II)

In the above formulas,

M is a dendrimer polylysine core;

T is a T cell epitope, preferably comprising peptide residues;

B _Pr is a protected carbohydrate B cell epitope comprising at least one carbohydrate moiety (b) wherein the hydroxyl group is protected by a Pr group,

The Pr group is allyl, p -methoxybenzyl (PMB), t -butyldimethylsilyl (TBDMS), benzyloxymethyl (BOM), levinyl (Lev), benzoyl (Bz), 2,5-difluorobenzoyl , Chloroacetyl, benzyl (Bn) or acetyl (Ac), or

Formed with two hydroxyl groups attached to C ₅ -C ₆ isopropylidene ketal or C ₅ -C ₆ cyclic alkylcarbonate

n is an integer indicating the number of -T-B groups covalently bonded to M.

In certain aspects, B _Pr is protected (α-GalNAc-Ser / Thr) ₃ .

In a further aspect, Pr is benzyl or acetyl.

In a further aspect, M is HO-βAla-K (K) ₂ .

In another aspect, T is QYIKANSKFIGITEL (SEQ ID N ° 1).

In another object, the present invention provides the use of a carbohydrate peptide conjugate M (TB _{Pr) n} for the preparation of a conjugate of M (TB) _n, M (TB _Pr ) _n and M (TB) _n are as defined above.

Other features of the invention will become apparent during the description of the following exemplary embodiments. These examples are presented for illustration of the invention and are not intended to limit them.

The method according to the invention advantageously minimizes byproduct synthesis, thereby improving the robustness of the process and scaling up the synthesis in a repeatable manner.

More specifically, protecting the hydroxyl group of the carbohydrate B cell epitope with an appropriate protecting group (Pr) before incorporating the B cell epitope into the M (T) _n building block provides better control of B cell epitope inclusion. Has been found to improve both yield and purity, particularly at large scale.

drawing
1: MAG-Tn3 synthesis according to protocol A of the method of the present invention. Molar equivalents are expressed in terms of amino groups. AA ^9-10 and AA ^15-16 were included as pseudo-proline dipeptides.
2: MAG-Tn3 synthesis according to protocol B of the method of the present invention. Molar equivalents are expressed in terms of amino groups. AA ^9-10 and AA ^15-16 were included as pseudo-proline dipeptides.

Example :

Example 1 : Preparation of MAG-Tn3 via Protocols A and B

General method

Synthesis of compound ( 6 ) was carried out stepwise using Fmoc chemistry on the solid phase. The protective group the amino acid side chains used was OtBu in Gln and Asn in Trt, Lys ^7, and in ¹¹ Boc Lys, Tyr, Ser and Thr tBu, Glu. In Lys ¹⁹ and Lys ²⁰ , the protecting group was Fmoc.

The net peptide content was determined by nitrogen analysis or quantitative amino acid analysis using Beckman 6300 analyzer after hydrolysis of the compound for 20 hours at 110 ° C. with 6N HCl.

HPLC / MS analysis was performed with an electrospray ionisation (positive mode) source (Waters) in an Alliance 2695 system combined with an ultraviolet detector 2487 (220 nm) and a Q-Tofmicro ^™ spectrometer (Micromass). Samples were cooled to 4 ° C. in an autosampler. A linear gradient was performed with acetonitrile + 0.025% FA (A) / purified water + 0.04% TFA + 0.05% FA (B) for 20 minutes. Columns were Zorbax 300SB C18 (3.5μ, 3 × 150 mm) (Agilent) (gradient 13-53% A) or XBridge ^™ BEH130 C18 (3.5μ, 2.1 × 150 mm) (Waters) (gradient 15-40% A). The temperature of the source was maintained at 120 ° C. and the desolvation temperature was 400 ° C. The cone voltage was 40V.

ESMS analysis was recorded in positive mode by direct infusion on the same mass spectrometer. Samples were dissolved at ˜5 μM concentration in purified water / acetonitrile (1/1) with 0.1% formic acid.

SELDI-TOF analysis was performed on a PCS 4000 mass spectrometer (Bio-Rad Labs). The H4 ProteinChip array surface was activated with 1 μL CH ₃ CN. Spots were incubated with reaction mixture (2.5 μL, 1 mg / mL) in a box for 20 min at RT. They were then washed with reaction buffer (3 × 1 min) and H ₂ O (3 × 1 min). To each spot was applied a matrix ( ₂ × 0.6 μL of sinapinic acid saturated in 50% CH ₃ CN / 0.5% TFA) and air dried. Spectra were generated with a laser set to ˜3 μJ from each array spot. The instrument was externally calibrated with the matrix and bovine ubiquitin, bovine cytochrome C, β-lactoglobulin with the settings described above.

The purity of compound ( 6 ) was analyzed by RP-HPLC using a 220 nm ultraviolet detector and an Agilent 1200 pump system. The column was Zorbax 300SB C18 (3.5μ, 3x150 mm) (Agilent) and the gradient was from 13% A to 53% A (0.8 mL / min, retention time 20.5 minutes) for 40 minutes acetonitrile + 0.1% TFA (A) / Purified water + 0.1% TFA (B).

Molar equivalents of all reagents are indicated for amino groups. The molar amount of protected intermediates ( 4 ) and ( 5 ) was calculated based on the starting material Fmoc-β-Ala-resin ( 1 ) substituents. The overall yield (table) includes all synthetic steps from compound ( 1 ). This was calculated from the net peptide content of the final product ( 6 ) from the Fmoc-β-Ala-resin ( 1 ) substituent.

Protocol A (Figure 1)

Fmoc-β-Ala-resin (low-substituted resin) ( One )

DMF in a round bottom flask with 2 g p -benzyloxybenzyl alcohol resin (Wang synthetic resin, 0.91 mmol / g, 100-200 mesh, polymer matrix: copoly (styrene-1% DVB), Novabiochem) (Applied Biosystems) was inflated for 1 hour. 311 mg (1 mmol) of dry Fmoc-β-Ala-OH (Novabiochem, Merck Chemicals Ltd) was dissolved in 8 mL of anhydrous CH ₂ Cl ₂ (Acros). Four drops of DMF were added to complete the dissolution. After addition of 77 μL (0.5 mmol) of DIC (Fluka), the reaction mixture was stirred under argon for 20 minutes at room temperature. The reaction mixture was evaporated until dry and a rotary evaporator was opened under argon. The residue was dissolved in minimal volume of DMF (6 mL) and the solution was added to the resin. 6 mg (0.05 mmol) of DMAP (Acros) dissolved in 0.5 mL of DMF was added and the suspension was gently stirred at room temperature for 2 hours.

According to the following procedure, the resin substitution ratio of the resin sample was measured by ultraviolet analysis. 2-6 mg of resin was transferred to a small sintered glass funnel with a Pasteur pipette. The resin was washed with DMF, CH ₂ Cl ₂ (Carlo Erba) and dried. The resin was transferred to a UV cell, precisely weighed, and then 2.8 mL of 20% piperidine (Aldrich) in DMF was added. The suspension was stirred with a Pasteur pipette for 2 minutes. Absorbance was read at 300.5 nm ( e = 7800) using a control cell containing 20% piperidine in DMF. The loading degree was found to be 0.1 mmol / g.

The resin was washed three times with DMF. Residual hydroxyl groups were capped using the following protocol. The resin was resuspended in 13 mL of DMF. 1.55 mL (16.4 mmol) of Ac ₂ O (Sigma) was added in 1 mL of DMF, followed by 660 mg (5.41 mmol) of DMAP in 1 mL of DMF. After gentle stirring at room temperature for 30 minutes, the suspension was filtered with a sintered glass funnel, washed three times successively with DMF, three times with CH ₂ Cl ₂ and then dried in a desiccator overnight. Resin ( 1 ) was stored at 4 ° C.

[QYIKANSKFIGITEL] ₄ -K ₂ -K-β-Ala-resin (protected peptide) ( 2 )

Tetravalent peptides were synthesized from 250 mg (25 μmol) of Fmoc-β-Ala-resin ( 1 ) in Applied Biosystems Peptide Synthesizer 433A using Fmoc chemistry. After each binding step, the Applied standard synthesis protocol was followed except for the additional wash step. Briefly, Fmoc groups were removed with 22% piperidine in NMP (Applied Biosystems) and deprotection was monitored by conductance. Lysine core uses HATU (Applied Biosystems) (1st cycle: 40eq, 2nd cycle: 20eq) / DIPEA (Applied Biosystems) (1st cycle: 80eq, 2nd cycle: 40eq) using binding reagent and NMP as solvent It consists of a continuous combination of two stages of Fmoc-Lys (Fmoc) -Applied Biosystems (OH) (40 eq, 2 eq: 20 eq). Is not necessary for this purpose, only due to the fact that the prepackaged cartridge is filled with 1 mmol of amino acids. Subsequent infusion of Fmoc-protected amino acids (Applied Biosystems, 10eq / amine) with standard side chain protecting groups was performed with HATU (10eq / amine) / DIPEA (20eq / amine) in NMP. AA in positions 15-16 and 9-10 are HATU (10 eq / amine) / DIPEA (20 eq / amine), respectively, Fmoc-Ile-Thr (Ψ ^{Me, Me} pro) -OH and Fmoc-Asn (Trt) -Ser ( ^{Me, Me} pro) -OH (10 eq / amine) (Novabiochem, Merck Chemicals Ltd).

[S (α-D-GalNAc (OBn) ₃ ) -T (α-D-GalNAc (OBn) ₃ ) -T (α-D-GalNAc (OBn) ₃ ) -QYIKANSKFIGITEL] ₄ -K ₂ -K-β-Ala ( 5 )

Starting from Compound ( 2 ) (25 μmol), glycosylated building blocks were manually included: Fmoc-T (α-D-GalNAc (OBn) ₃ ) -Ficher Chemicals AG (First Cycle), Fmoc -T (α-D-GalNAc (OBn) ₃ ) -OH (second cycle) and Fmoc-S (α-D-GalNAc (OBn) ₃ ) -OH (Ficher Chemicals AG) (third cycle). Briefly, dry building blocks (0.3 mmol, 3eq / free amino group) were dissolved in a minimal amount of DMF (˜2 mL). A solution of 55 mg (0.145 mmol) of HATU (Novabiochem) in 0.5 mL DMF was added and the resulting mixture was added to the resin. After addition of 52 μL (0.3 mmol) of DIPEA (Aldrich), the suspension was mechanically stirred. Three binding steps were monitored by Kaiser test [1] and were completed at 1h, 1h and 1h, respectively. After each binding step, the resin was washed with DMF (4 times). All Fmoc cleavage was performed with 20% piperidine treatment in DMF of the resin. After each deprotection, the resin was washed successively with DMF (6 times), CH ₂ Cl ₂ (6 times), and DMF (6 times). At the end of the synthesis, the resin was washed extensively with DMF and CH ₂ Cl ₂ and dried in a desiccator. 10 mL of Applied Biosystems (TFA) / purified water / TIS (Acros) (95 / 2.5 / 2.5 v / v / v) was added to the resin at 4 ° C. and the mixture was stirred for 1 hour 30 minutes at room temperature. After filtration of the resin, the solution was concentrated and the crude product was precipitated with diethyl ether. After centrifugation, the pellet was dissolved in purified water and lyophilized to give 229 mg of crude glycopeptide ( 5 ).

[S (α-D-GalNAc) -T (α-D-GalNAc) -T (α-D-GalNAc) -QYIKANSKFIGITEL] ₄ -K ₂ -K-β-Ala or MAG-Tn3 ( 6 )

Starting from compound ( 5 ) with TfOH [2-4],

At room temperature, 200 mg (0.014 mmol) of Compound ( 5 ) was dissolved in 2.96 mL of TFA, 1.78 mL of DMS (Sigma-Aldrich), and 587 μL of Metacresol (Sigma-Aldrich). The solution was cooled to -10 ° C and 587 μL of TfOH (Fluka) was added and the mixture was then -10 ° C (TfOH / TFA / DMS / m -cresol 1/5/3/1 v / v / v / for 1 hour 15 minutes). v). The product was precipitated with diethyl ether, and after centrifugation, the pellet was dissolved in purified water and lyophilized to give 372 mg of crude glycopeptide. The product was dissolved in 7.7 mL of 0.05 M ammonium acetate buffer and the pH was adjusted to 7 with 1 M ammonia. After 1 hour at room temperature, the solution was lyophilized to yield 412 mg of crude product. The product was purified by RP-HPLC using a 230 nm ultraviolet detector and an Agilent 1200 pump system. The column was Zorbax C18 (5μ, 300 μs, 9.4 × 250 mm) (Agilent) and gradient was performed for 20 minutes from 73/27 to 60/40 with purified water (0.1% TFA) / acetonitrile. Purification resulted in 3.9 mg (net peptide content) of compound ( 6 ) having a purity of 95.90%. Overall yield was 1.6%.

Protocol B (Figure 2)

[QYIKANSKFIGITEL] ₄ -K ₂ -K-β-Ala-resin (protected peptide) ( 2 )

Tetravalent peptides were extracted from 36.9 g (4.8 mmol) of Fmoc-β-Ala-Tentagel R Trt resin ( 1 ) (0.13 mmol / g) (Rapp Polymere) in a manual peptide synthesizer equipped with a Schmizo reactor until Tyr ⁵ was included. Synthesized. Prior to the elongation process, the resin was expanded in DMF for 2-3 hours and washed with 240 mL of DMF (3 times, 2 minutes / cycle). After each binding, Fmoc groups were removed with 20% piperidine in 240 mL of DMF (3 steps, 20 minutes each). In the case of Glu ¹⁷ , Asn ⁹ and Gln ⁴ , 2% HOBt was added to the deprotection solution. After each deprotection, the resin was subjected to 240 mL of DMF (4 times, 2 minutes / cycle), 240% L 2% HOBt in DMF (2 times, 5 minutes / cycle), and 240 mL of DMF (2 times, 2 minutes / cycle). Washing in succession).

Amino acid binding (1.5-2 eq / amine) was performed with DIC / HOBT (1.5-2 eq / amine, respectively) at room temperature in DMF (111 mL) (see details below). AA at positions 15-16 and 9-10 were included as Fmoc-Ile-Thr (Ψ ^{Me, Me} pro) -OH and Fmoc-Asn (Trt) -Ser (Ψ ^{Me, Me} pro) -OH, respectively. After 30 minutes, a fresh portion of DIC (1.5-2eq) was added to the reaction mixture. The binding step was monitored by Kaiser test [1]. After Leu ¹⁸ to Ser ¹ , binding for 1 hour with DIC / HOBT (in the same amount), PyBOP reagent was added (see details below) and the pH was adjusted to 7 × by dropwise addition of DIPEA. After 30 minutes, the resin was washed with 240 mL of DMF (5 times, 2 minutes / cycle) and an acetylation step was performed from Leu ¹⁸ to Thr ² . Acetylation was performed with acetic anhydride (1 eq / amine) in 111 mL DMF in the presence of pyridine (1 eq / amine) at room temperature. After 20 minutes, the resin was washed with 240 mL DMF (6 times, 2 minutes / cycle). After the incorporation of Tyr ⁵ , the resin was washed extensively with 240 mL of DMF (8 times, 2 minutes / cycle) and 240 mL of CH ₂ Cl ₂ (8 times, 2 minutes / cycle).

After the incorporation of Tyr ⁵ , assembly was carried out using the same protocol on the 0.15 mmol or 4.65 mmol scale and peptide-resin ( 2 ) was provided for compound ( 4 ) and compound ( 5 ) respectively.

[S (α-D-GalNAc (OAc) ₃ ) -T (α-D-GalNAc (OAc) ₃ ) -T (α-D-GalNAc (OAc) ₃ ) -QYIKANSKFIGITEL] ₄ -K ₂ -K-β-Ala ( 4 )

With respect to the compound (2) was carried out the synthesis from Compound (2) (0.15mmol) as described above. The binding step was performed using the following AA building blocks [5] and reagents.

At the end of the synthesis, glycopeptide resin (0.15 mmol) is suspended in TFA / TIS / H ₂ O (95 / 2.5 / 2.5 v / v / v) (10 mL / g of glycopeptide resin) and 20 ° C. ± 1 h. Stir at 2 ° C. After filtration, the resin was washed twice with the same TFA mixture (2 mL / g glycopeptide resin per wash). The filtrate and washes were combined and stirred for an additional 30 minutes at 20 ° C ± 2 ° C. After concentration (bath temperature ≦ 35 ° C.), the crude product was precipitated with DIPE (glycopeptide resin ˜35 mL / g). After filtration and washing with DIPE, the solid was dried (t ° ≦ 30 ° C.) to give 750 mg of crude compound ( 4 ).

ESMS: 12409.589 (C ₅₅₃ H ₈₅₅ N ₁₀₇ O ₂₁₃ calculated (calcd) 12410,465)

[S (α-D-GalNAc (OBn) ₃ ) -T (α-D-GalNAc (OBn) ₃ ) -T (α-D-GalNAc (OBn) ₃ ) -QYIKANSKFIGITEL] ₄ -K ₂ -K-β-Ala ( 5 )

With respect to the compound (2) was carried out the synthesis from Compound (2) (4.65mmol) as described above. The binding step was performed using the following AA building blocks (Ficher Chemicals AG) and reagents. At the end of the synthesis, 84.87 g of glycopeptide resin was obtained.

Compound ( 4 ) was treated with glycopeptide resin (20 g, 1.096 mmol) as described above to yield 9.95 g of crude compound ( 5 ).

ESMS: 14141.433 (C ₇₃₃ H ₉₉₉ N ₁₀₇ O ₁₇₇ Output 14141,610)

[Reference]: This protocol gave a crude compound equivalent to that obtained according to Protocol A, namely from Fmoc-β-Ala- p -benzyloxybenzyl alcohol resin (royal synthetic resin) (see above).

[S (α-D-GalNAc (OH) ₃ ) -T (α-D-GalNAc (OH) ₃ ) -T (α-D-GalNAc (OH) ₃ ) -QYIKANSKFIGITEL] ₄ -K ₂ -K-β-Ala or MAG-Tn3 ( 6 )

Starting from compound ( 4 ) with hydrazine [7],

100 mg (20 μmol) of Compound ( 4 ) were dissolved in 3.2 mL of MeOH. 567 μL (11.3 mmol) of hydrazine monohydrate was added and the solution was stirred at room temperature. After 2 hours 30 minutes, the solution was cooled to 0 ° C. and 3.2 mL of acetone was added. After 1 hour, the solution was concentrated and co-distilled five times with acetone. The crude glycopeptide was lyophilized to yield 117 mg. The product was purified by RP-HPLC using an Agilent 1200 pump system with an ultraviolet detector at 230 nm. The column was Zorbax C18 (5μ, 300 μs, 9.4 × 250 mm) (Agilent) and gradient was performed for 20 minutes with purified water (0.1% TFA) / acetonitrile from 72/28 to 62/38. The purification gave 5.8 mg (net peptide content) of 96.4% purity ( 6 ). Overall yield was 2.7%.

Starting from compound ( 4 ) with MeONa,

24 mg (4.8 μmol) of Compound ( 4 ) was dissolved in 3.2 mL of MeOH. The pH was adjusted to 14 with 1% MeONa in MeOH (pH meter, moist pH paper ˜10.5) and the solution was stirred at RT. The reaction was monitored by RP-HPLC. After 2 hours, the reaction was neutralized with dry ice and evaporated to dryness. The crude peptide was dissolved in 1% aqueous TFA and lyophilized. The product was purified by RP-HPLC using an Agilent 1200 pump system with an ultraviolet detector at 230 nm. The column was Kromasil C4 (5μ, 100 μs, 10 × 250 mm) (AIT) and gradient was performed for 30 minutes with 0.1% aqueous TFA (VWR) / acetonitrile (Carlo Erba) from 73/27 to 62/38. The purification gave 754 µg (net peptide content) of Compound ( 6 ) having a purity of 91.4%. Overall yield was 1.4%.

Starting from compound ( 5 ) with H ₂ ,

10 g (1.1 mmol) of Compound ( 5 ) were dissolved in 800 mL of NMP / H ₂ O 87.5 / 12.5. After adding 4 g of 10% Pd / C type 39 (Johnson Matthey), the reaction was stirred at 37 ° C. and 5 bar for 170 hours. Two additional amounts of catalyst were added portionwise after 72 hours (2 g) and 120 hours (2 g). At the end of the reaction, the catalyst was filtered through celite and washed with NMP / H ₂ O 87.5 / 12.5. The resulting filtrate was collected with other filtrates from a similar reaction (total 1.35 mmol). After dilution with H ₂ O (until NMP / H ₂ O 10/90), the filtrate was purified in two steps by RP-HPLC. First purification was performed on a Vydac C18 column (300 mm 3, 10-15 μm, 50 mL / mn) with TFA / H ₂ O / CH ₃ CN 0.1 / 94.9 / 5.0 v / v / v (A) and TFA / H ₂ O / CH ₃ CN 0.1 / 49.9 / 50.0 v / v / v (B). The gradient was 0% B for 15 minutes, 0-40% B for 5 minutes and 40-80% B for 60 minutes. Second purification was carried out on a Vydac C18 column (300 μs, 10-15 μm, 50 mL / mn) with AcOH / H ₂ O / CH ₃ CN 0.5 / 94.5 / 5.0 v / v / v (A) and AcOH / H ₂ O / CH ₃ CN 0.5 / 49.5 / 50.0 v / v / v (B). The gradient was 0% B for 15 minutes, 0-20% B for 5 minutes and 20-60% B for 60 minutes. After concentration on a Daisogel SP-300-10-ODS-AP column (20 mL / mn, isocratic TFA / H ₂ O / CH ₃ CN 0.1 / 49.9 / 50.0 v / v / v) by RP-HPLC, the solution is spun. Evaporation and lyophilization on an evaporator yielded 225 mg (net peptide content) of compound ( 6 ) with a purity of 95.3%. Overall yield was 1.5%.

ESMS: 10897.387 (C ₄₈₁ H ₇₈₃ N ₁₀₇ O ₁₇₇ Output 10897,123)

conclusion

The results obtained are summarized in the table below:

¹ Refs. 5 and 9, (WO 9843677 multi-antigen glycopeptide carbohydrates, vaccines comprising the same and uses thereof, Ref. 11)

Calculated on net peptide content from ² Fmoc-βAla-resin substitutions (includes all synthetic steps from Compound ( 1 ))

Measured by ³ RP-HPLC: column Zobrax 300SB C18 (3.5 μ, ³ × 150 mm) (Agilent), 0.8 mL / min, A: acetonitrile + 0.1% TFA, B: purified water + 0.1% TFA, 13 for 40 minutes Gradient of A from% to 53%, detected at 220 nm.

^{4 on the} final product (net peptide content).

Compared to the first process using an unprotected carbohydrate synthon (FIGS. 1 and 2), the novel process (with protected carbohydrates, II, 1 and 2) allows:

-Minimize synthetic byproducts

Improved process repeatability

Scale up synthesis in a repeatable manner

Among the tested protocols of the novel process (Tables, Figures 1 and 2), three emerge as the best strategy: Ac / hydrazine, Bn / TfOH and Bn / H ₂ (protector / deprotection method) (Table). All of these gave MAG-Tn3 with a purity ≧ 95% in a repeatable manner.

The Bn / TfOH method provided MAG-Tn3 with 1.6% overall yield. This method requires a compromise between complete deprotection and degradation. Alternatively, the Bn / H ₂ method provides MAG-Tn 3 in similar yield (1.5%), and most importantly, the process was certified on the 225 mg scale. Finally the Ac / hydrazine method gave the highest overall yield (2.7% compared to 1.6% and 1.5%).

MAG-Tn3 based on another peptide (PV = KLFAVWKITYKDT) (SEQ ID N ° 4) was also prepared according to the method of the present invention (Ac / hydrazine, Bn / TfOH).

Example 2 Influence of Resin Substitution Rate and Characteristics of Fixed Phase Used in Purification of MAG-Tn3

MAG-Tn3 from polystyrene resins (sold under the trade name of Fmoc-β-Ala-TentaGel R Trt) functionalized with Fmoc-β-Ala according to Protocol B, with two different substitution ratios: 0.13 or 0.1 mmol / g (ie Number of groups grafted with Fmoc-β-Ala to the weight of unrafted resin).

Purification of crude MAG-Tn3 was performed by RP-HPLC on three separate stationary phases (reverse phases) based on octadecyl group grafted silica gel, ie Vydac®, Jupiter® and Daisogel®. First purification to TFA / H ₂ O / CH ₃ CN 0.1 / 94.9 / 5.0 v / v / v (A) and TFA / H ₂ O / CH ₃ CN 0.1 / 49.9 / 50.0 v / v / v (B) Was performed. The gradient was 0% B for 15 minutes, 0-40% B for 5 minutes and 40-80% B for 60 minutes. Second purification to AcOH / H ₂ O / CH ₃ CN 0.5 / 94.5 / 5.0 v / v / v (A) and AcOH / H ₂ O / CH ₃ CN 0.5 / 49.5 / 50.0 v / v / v (B) Was performed. The gradient was 0% B for 15 minutes, 0-20% B for 5 minutes and 20-60% B for 60 minutes.

The results are reported in the table below.

^a powder weight

^b RP-HPLC Zorbax 300SB C18 (3.5μ, 3 × 150 mm, Agilent), A: acetonitrile + 0.1% TFA, B: H ₂ O + 0.1% TFA, 15-53% A (40 minutes).

^c Yield was calculated from the net peptide content of the final product, including all synthetic steps from Fmoc-β-Ala-resin.

Vydac ^® C18, 300 μs, 10-15 μm (Grace), ref 218 MSB1015 or 218TPB1015 or 238TPB1015

Jupiter ^® C18, 300 μs, 10 μm (Phenomenex), ref 04G-4055

Daisogel ^® C18, 300 Å, 10 μm (Daiso), ref SP-300-10-ODS-RPS

These results demonstrate that both the process yield of the conjugate preparation according to the invention, and the purity of the conjugate obtained, can be highly improved by reducing and / or reducing the substitution rate of the resin and / or using a suitable stationary phase.

Abbreviation
AA amino acids
Ac acetyl
AcOH acetic acid
Bn benzyl
Boc tert-butoxycarbonyl
tBu tert-butyl
DIC N, N'-diisopropylcarbodiimide
DIPEA diisopropylethylamine
DIPE diisopropyl ether
DMAP 4-dimethylaminopyridine
DMF Dimethylformamide
DMS dimethyl sulfide
DVB divinylbenzene
EtOH Ethanol
FA formic acid
Fmoc 9-fluorenylmethoxycarbonyl
HATU 2- (1H-9-Azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate
HOBt N-hydroxybenzotriazole
HPLC / MS High Performance Liquid Chromatography / Mass Spectrometry
MAG Multiple Antigen Glycopeptide
MeOH Methanol
MeONa sodium methylate
ESMS electrospray mass spectrometry
MW molecular weight
NMP N-methylpyrrolidone
NMR nuclear magnetic resonance
PyBOP Benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate
RP-HPLC Reversed Phase High Performance Liquid Chromatography
RT room temperature
SELDI-TOF MS surface-enhanced laser desorption / ionization time-of-flight mass spectrometry
TBS tert-butyldimethylsilyl
TFA trifluoroacetic acid
TfOH trifluoromethanesulfonic acid
THF tetrahydrofuran
TIS triisopropylsilane
TMSBr trimethylsilyl bromide
Trt trityl

                         SEQUENCE LISTING <110> Institut Pasteur   <120> METHOD FOR PREPARING MULTIPLE ANTIGEN GLYCOPEPTIDE CARBOHYDRATE        CONJUGATES <130> F 226 CAS 188 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Tetanus toxin fragment <400> 1 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu 1 5 10 15 <210> 2 <211> 21 <212> PRT <213> Artificial sequence <220> <223> Tetanus toxin fragment <400> 2 Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Val Lys Val Ser 1 5 10 15 Ala Ser His Leu Glu             20 <210> 3 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Tetanus toxin fragment <400> 3 Gly Gln Ile Gly Asn Asp Pro Asn Arg Asp Ile Leu 1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial sequence <220> Poliovirus VP1 protein fragment <400> 4 Lys Leu Phe Ala Val Trp Lys Ile Thr Tyr Lys Asp Thr 1 5 10

Claims (24)

As a method of preparing a carbohydrate T cell epitope conjugate of formula (I):
M (TB) _n (I)
In the above formula:
M is a dendrimer polylysine core;
T is a T cell epitope;
B is a carbohydrate B cell epitope comprising at least one carbohydrate residue (b);
n is an integer representing the number of -TB groups covalently bonded to M;
A method comprising the following steps:
i) A protected carbohydrate B cell epitope (B _Pr ), wherein the hydroxyl group of the carbohydrate residue (b) is protected with a protecting group (Pr), is bound to compound M (T) _n to give a carbohydrate T cell epitope conjugate M (TB _Pr ) _n , wherein the protecting group (Pr) is allyl, p -methoxybenzyl (PMB), t-butyldimethylsilyl (TBDMS), benzyloxymethyl (BOM), levulinyl (Lev) , benzoyl (Bz), 2,5-difluoro-benzoyl, chloroacetyl, benzyl (Bn) or or selected from the group consisting of acetyl (Ac), C ₅ -C ₆ isopropylidene ketal or a C ₅ -C ₆ between Formed with two hydroxyl groups attached to the click alkylcarbonate;
And
ii) removing the protecting group Pr from the obtained conjugate M (TB _Pr ) _n to obtain a carbohydrate T cell epitope conjugate M (TB) _n . The process of claim 1, wherein M (TB) _n is selected from formula (Ia) or (Ib):

In the above formula, K is a lysine residue,
T and B are as defined in claim 1. The method of claim 1 or 2, wherein M is (K) ₂ K-βAla-OH of the formula:

The method of claim 1, wherein T is a T cell epitope comprising a peptide. The method of claim 4, wherein T is or comprises a CD8 ⁺ or CD4 ⁺ T cell epitope. The method of claim 5, wherein T is or comprises a CD4 ⁺ T cell epitope. The method of claim 6, wherein T is or comprises a poliovirus (PV) fragment protein, tetanus toxin fragment, or PADRE peptide. 8. The method of claim 7, wherein T is a peptide consisting of QYIKANSKFIGITEL (SEQ ID No 1). 9. The method of claim 1, wherein the carbohydrate moiety (b) is attached to an amino acid, peptide, or lipid moiety. 10. The method of claim 1, wherein B is or comprises a carbohydrate moiety selected from the group consisting of:
α-GalNAc-Ser,
α-GalNAc-Thr,
β-GalNAc-Ser,
β-GalNAc-Thr,
β-Gal- (1-3) -α-GalNAc-Ser,
β-Gal- (1-3) -α-GalNAc-Thr,
(α-GalNAc-Ser / Thr) ₂ ,
(α-GalNAc-Ser / Thr) ₃ , and
-(α-GalNAc-Ser / Thr) ₆ . The method of claim 10, wherein B is or comprises residues (α-GalNAc-Ser / Thr) ₃ . The method according to any one of claims 1 to 11, wherein the protecting group Pr is benzyl (Bn) or acetyl (Ac). The method according to any one of claims 1 to 12, wherein step i) comprises the following steps:
a) combining a first protected carbohydrate moiety (b _Pr ) with compound M (T) _n , _{wherein the} hydroxyl group is protected by a protecting group (Pr) as defined in any one of claims 1-12. To form a carbohydrate T cell epitope conjugate M (Tb _Pr ) _n ; And
b) repeating step a) until a further protected carbohydrate moiety (b _Pr ) gives a protected carbohydrate conjugate M (TB _Pr ) _n of formula (II). The method of claim 1, wherein Pr is benzyl. The method of claim 14, wherein the benzyl group is removed in the presence of TfOH or H ₂ . The method according to any one of claims 1 to 13, wherein Pr is acetyl. The method of claim 16, wherein the acetyl group is removed in the presence of hydrazine or MeONa. As a carbohydrate T cell epitope conjugate of formula (II):
M (TB _Pr ) _n (II)
In the above formulas,
M is a dendrimer polylysine core;
T is a T cell epitope;
B _Pr is a protected carbohydrate B cell epitope comprising at least one carbohydrate moiety (b) in which the hydroxyl group is protected by a Pr group as defined in claim 1;
carbohydrate T cell epitope conjugates wherein n is an integer representing the number of -TB groups covalently bound to M. 19. The carbohydrate T cell epitope conjugate according to claim 18, wherein T is or comprises a peptide. The carbohydrate T cell epitope conjugate according to claim 18 or 19, wherein Pr is benzyl (Bn) or acetyl (Ac). The carbohydrate T cell epitope conjugate according to any one of claims 18 to 20, wherein B _Pr is protected (α-GalNAc-Ser / Thr) ₃ . The carbohydrate T cell epitope conjugate according to any one of claims 18 to 21, wherein M is HO-βAla-K (K) ₂ . The carbohydrate T cell epitope conjugate according to any one of claims 18 to 22, wherein T is QYIKANSKFIGITEL (SEQ ID N ° 1). The carbohydrate T cell epitope conjugate M according to any one of claims 18 to 23 for preparing the carbohydrate peptide conjugate M (TB) _n as defined in any one of claims 1 to 17. _Pr ) _n uses.

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